Undesirable Off-target Effects Research Articles

P53 activation by knockdown technologies

Morpholino phosphorodiamidate antisense oligonucleotides (MOs) and short interfering RNAs (siRNAs) are commonly used platforms to study gene function by sequence-specific knockdown. Both technologies, however, can elicit undesirable off-target effects. We have used several model genes to study these effects in detail in the zebrafish, Danio rerio. Using the zebrafish embryo as a template, correct and mistargeting effects are readily discernible through direct comparison of MO-injected animals with well-studied mutants. We show here indistinguishable off-targeting effects for both maternal and zygotic mRNAs and for both translational and splice-site targeting MOs. The major off-targeting effect is mediated through p53 activation, as detected through the transferase-mediated dUTP nick end labeling assay, acridine orange, and p21 transcriptional activation assays. Concurrent knockdown of p53 specifically ameliorates the cell death induced by MO off-targeting. Importantly, reversal of p53-dependent cell death by p53 knockdown does not affect specific loss of gene function, such as the cell death caused by loss of function of chordin. Interestingly, quantitative reverse-transcriptase PCR, microarrays and whole-mount in situ hybridization assays show that MO off-targeting effects are accompanied by diagnostic transcription of an N-terminal truncated p53 isoform that uses a recently recognized internal p53 promoter. We show here that MO off-targeting results in induction of a p53-dependent cell death pathway. p53 activation has also recently been shown to be an unspecified off-target effect of siRNAs. Both commonly used knockdown technologies can thus induce secondary but sequence-specific p53 activation. p53 inhibition could potentially be applicable to other systems to suppress off-target effects caused by other knockdown technologies.

FIELD EVALUATION OF INSECTICIDES FOR THE CONTROL OF THE EUROPEAN SKIPPER, THYMELICUS LINEOLA (LEPIDOPTERA: HESPERIIDAE), IN QUEBEC

AbstractThe efficacy of seven chemical insecticides, five commercial preparations of Bacillus thuringiensis, and two preparations containing diatom dust were tested against the European skipper, Thymelicus lineola (Ochs.), under field conditions. Satisfactory control was obtained with carbaryl, carbofuran, methomyl, permethrin, trichlorfon, Dipel 45B(=Dipel SC), Dipel WP, Thuricide 26B, Thuricide HPC, and Novabac 3 while dimethoate, chlorpyrifos, and the diatom dusts afforded little or no foliage protection. While larval mortality occurred less rapidly when B. thuringiensis preparations were applied, their use is strongly recommended as they afford the same degree of foliage protection as chemical insecticides and diminish possible undesirable off-target effects.

BACILLUS THURINGIENSIS AS A MEANS OF CONTROL FOR THE EUROPEAN SKIPPER, THYMELICUS LINEOLA (LEPIDOPTERA: HESPERIIDAE)

To date no entirely satisfactory control method has been developed for the European skipper, Thymelicus lineola (Ochs.), which was first introduced into North America in London, Ont., around 1910 (Saunders 1916) and has since spread throughout much of Canada and northern United States (Arthur 1966; Burns 1966; McNeil et al. 1975). In Quebec this insect has reached epidemic proportions in several areas (McNeil et al. 1975) and while Sevin® has been utilized, its application causes certain undesirable off-target effects. It is therefore essential to develop a means of control which does not cause these ecological problems.